Abstract Wnt3a is an important secreted protein of the canonical Wnt pathway and its activation can promote cell proliferation and division. In order to reveal the roles of Wnt3a in regulating the proliferation and differentiation towards insulin-secreting cells of porcine pancreatic stem cells (PSC), the full length of Wnt3a cDNA fragment was amplified from pGKS2P(+)-Wnt3a plasmid by RT-PCR. The recombinant plasmid pIRES2-AcGFP1-Wnt3a was constructed by inserting Wnt3a fragment sequenced correctly into eukaryotic expression vector pIRES2-AcGFP1. After identifying with double digestion of EcoRⅠ/BamHⅠ, we transfected pIRES2-AcGFP1-Wnt3a into porcine PSC by lipofectamin 2000. We obtained the stably transfected pocine PSC strain after screening culture by G418 for 3 weeks. Almost all cells expressing green fluorescence protein were observed by fluorescence microscope. The expression of Wnt3a mRNA and protein obviously increased in stable transfection group of Wnt3a compared to control group when detected by RT-PCR and Western blot. These results indicated that we successfully established the porcine PSC strain stably expressing Wnt3a. This study provide basal data for further exploring the mechanism of Wnt3a in porcine PSC proliferation and differentiation.
